Stem-regulated, plant defense promoter and uses thereof in tissue-specific expression in monocots

ABSTRACT

The invention is directed to isolated promoters from stem-regulated, defense-inducible genes, such as JAS promoters. The promoters are useful in expression cassettes and expression vectors for the transformation of plants. Particularly, the invention provides transgenic plants of rice and sugarcane that have been modified such that expression of a heterologous coding sequence is directed by an JAS promoter and is limited to stem tissues or may be upregulated by the presence of a defense-inducing agent. The invention also discloses methods for producing the expression vectors and transgenic plants.

PRIORITY CLAIM

The present invention claims priority under 35 U.S.C. §119(e) to U.S.Provisional Pat. Appln. Ser. No. 60/437,974 filed on Jan. 3, 2003 andtitled “Stem-Expressed Promoter for Tissue-Specific Expression inMonocots”.

FIELD OF THE INVENTION

The present invention relates to the fields of plant functionalgenomics, molecular biology and genetic engineering, and specifically toselective regulation of gene expression in plants. In particular, thepresent invention relates to novel isolated nucleic acids havingpromoters capable of conferring stem-regulated expression and operablein plant defense. The promoters may be used in monocots, includingpolyploid species, to direct stem-regulated or defense-inducibleexpression of a heterologous gene.

BACKGROUND

A critical component of agriculture biotechnology is the use of highlyregulated promoters to express agronomically important genes in cropplants so that genes of interest are expressed at optimal levels inappropriate tissues.

Monocots form a substantial portion of the world's food supply.Sugarcane is considered as one of the major worldwide crop for sugarsupply with a net value of $20 billion per year. This crop could benefitfrom biotechnology approaches to engineer plants for disease, pest andherbicide resistance as well as improved sugar yield. However, comparedto other major crops, there has been little research in developingsugarcane specific technologies such as genes and promoters that arefunctional in sugarcane. At the present time, there are no public-domainsugarcane promoters available for use in sugarcane transformation.

The selection of a promoter is often a critical factor in determiningwhen and where within the plant the gene of interest is expressed.

A number of gene promoters that drive high levels of constitutivetransgene expression are available for monocot plants. These include themaize ubiquitin promoter (Quail et al., 1996, U.S. Pat. No. 5,510,474),the rice actin1 promoter (McElroy and Wu, 1997, U.S. Pat. No.5,641,876), various enhanced cauliflower mosaic virus (CaMV) 35Spromoters (Mitsuhara et al., 1996, Plant Cell Physiol. 37:49–59) andpromoters isolated from banana streak virus (Schenk et al., 2001, PlantMol. Biol. 47:399–412). Promoters that have been isolated so far fromsugarcane correspond to two polyubiquitin genes and confer constitutivegene expression in non-host plants such as rice (Wei et al., 1999, J.Plant Physiol. 155:513–519; Wei et al., 2003, J. Plant Physiol.160:1241–1251).

Constitutive promoters may be suitable for the production of a desiredprotein in large quantities in all tissues of the plant throughoutdevelopment. The energy requirements for high level constitutiveproduction of the protein are often so great that other normal plantgrowth processes are compromised. For example, expression of a proteinin a non-tissue specific manner as directed by a constitutively activepromoter has often resulted in slow-growing or dwarfed plants. Eventhough providing constitutive expression of a gene is often desirable,it is also desirable in many instances to direct high expression of agene to particular tissues and/or time of development in a plant.Tissue-specific promoters are capable of selectively expressing anintroduced gene in desired tissues. Tissue-specific promoters may alsobe inducible, e.g. activated by internal or external agents such asphytohormones or defense inducing agents.

Restricting expression of the target protein to a particular tissue ororgan or to specific events triggered when the plant is challenged withan external agent may be desirable to minimize possible toxic effects ofsome ‘agronomic’ gene products and to optimize overall plant growth andproduction. Furthermore, it is increasingly clear that promoter functionvaries from species to species. Thus it is essential to have promoterswhich are expressed specifically in target tissues of specific plants inorder to genetically engineer plants.

Several promoters are currently being used for tissue-specific,heterologous gene expression in monocots. For example, the promoterregions from genes coding for hydrolases (β-glucanase), cysteineprotease inhibitors (cystatin-1) or glucosidases α-glucosidase) havebeen used to direct germination-specific expression of a heterologousDNA sequence in transgenic barley cells and kernels (Wolf, 1992, Mol.Gen. Genet. 234:33–42; Jensen et al., 1996, Proc. Natl. Acad. Sci. USA93:3487–3491; Mikkonen et al., 1996, Plant Mol. Biol. 31:239–254; Jensenet al., 1998, U.S. Pat. No. 5,712,112; Lok et al., 2002, U.S. Pat. No.6,359,196). The promoter for the glutamine synthetase gene has been alsoused to drive tissue-specific expression within the developing kernelsof transgenic maize plants (Muhitch et al., 2002, Plant Science163:865–872). Xylem- and phloem-specific promoters that are active inrice have also been reported, including the rice tungro baciliform virusand peroxidase gene promoters (Yin et al., 1997, Plant Journal12:1178–1188; Ito et al., 2000, Plant Science 155:85–100); however it isnot clear whether these promoters are active in other monocots, such asmaize, sorghum and sugarcane. Furthermore, none of the above reportedpromoters are stem-regulated, which may be significant for crops such assugarcane where the stem holds a large portion of the commercial valueof the plant.

SUMMARY OF THE INVENTION

The present invention provides regulatory sequences which directstem-regulated or defense-inducible expression and include novelpromoters from stem-expressed defense-inducible gene designatedjasmonate-induced protein (JAS). The subject promoters may have specificadvantages over the currently available tissue-specific promoters intheir enhanced specificity in regulating gene expression in stem tissuesand in response to induction by external stimuli such as plantdefense-inducing agents. The subject promoters may be very useful instrategies aimed at altering carbon metabolism in the sucroseaccumulating tissues, and for driving expression of insecticidalproteins in sugarcane. These promoters may also be applied to thedevelopment of improved pest and disease tolerant rice plants.

The present invention is directed to isolated nucleic acids includingpromoters operable primarily in the stem or in response to stimulationby defense-inducing agents. The subject promoters hybridize understringent conditions to a promoter isolated from sugarcane, designatedthe JAS promoter, which promoter has the nucleotide sequence as setforth in SEQ ID NO:1

In other embodiments of the invention, the subject isolated nucleicacids include promoters which direct stem-regulated or defense-inducibleexpression and have a sequence identity (sequence similarity) of fromabout 60% to about 65%, from about 65% to about 75%, from about 75% toabout 85%, or from about 85% to about 90% when compared to thenucleotide sequence of the JAS promoter as set forth in SEQ ID NO: 1.Promoters of the present invention may also have a sequence identity ofat least 60%, at least 70%, at least 80%, at least 90%, at least 95%, orat least 98% when compared to the JAS promoter of SEQ ID NO: 1. It willbe understood by one skilled in the art that where the designation “JASpromoter” is used in the present specification, use of other nucleicacids having hybridization characteristics or homologous sequences asset forth above may be appropriate as well unless a specific identity orsequence is clearly indicated. However, all JAS promoters retain someability to direct stem-regulated transcription or defense-inducibletranscription.

In a further embodiment, the present invention is directed to anisolated nucleic acid having a promoter which directs stem-regulated ordefense-inducible expression, which promoter has the nucleotide sequenceas set forth in SEQ ID NO:1.

Other embodiments of the invention include expression vectors includingan isolated nucleic acid having a promoter which directs stem-regulatedor defense-inducible expression, including an JAS promoter. Theexpression vector may, more specifically, be transferred into a plantcell to be transformed in such a manner as to allow expression of anencoded protein in tissues derived from the plant cell. The vector mayalso transmitted into the plant cell in such a manner as to allowinheritance of the nucleic acid into the second progeny of plantsgenerated from a plant derived from the transformed plant cell. Morespecifically, such inheritance may be Mendelian. Examples of planttransformation expression vectors including the JAS promoter are shownin FIGS. 2 and 3.

In still another embodiment, the present invention may be directed tocells, tissues and plants transformed with an expression vectorincluding an isolated nucleic acid having a promoter which directsstem-regulated or defense-inducible expression, including an JASpromoter, and the progeny generated from such transformed plants. Inspecific embodiments, the plant may be a monocot, such as maize, rice,sugarcane or sorghum.

In still further embodiments, the present invention provides anexpression cassette which includes an isolated nucleic acid having apromoter which directs stem-regulated or defense-inducible expressionincluding an JAS promoter operably linked to a heterologous gene or anucleic acid encoding a sequence complementary to the native plant geneand vectors containing such expression cassettes.

The present invention also includes methods for directing stem-regulatedexpression in a tissue or plant by providing such tissue or plant withan isolated nucleic acid having a promoter including an JAS promoter toeffect such stem-regulated expression. Other methods relate to directingdefense-inducible expression in a plant by providing such plant with anisolated nucleic acid having a promoter including an JAS promoter toeffect such defense-inducible expression.

Finally, the invention includes methods of isolating a tissue-specificpromoter in polyploid monocots by first selecting potentialtissue-specific cDNA clones using differential hybridization analysisfollowed by microarray analysis if the cDNA clones using probes derivesfrom total RNA of various tissues. This may be followed by furtherconfirmation techniques such as real time PCR and RNA gel blot analysisusing total RNA of various tissues.

BRIEF DESCRIPTION OF THE DRAWINGS

Specific embodiments of the present invention are further described inthe following detailed description taken in conjunction with theaccompanying drawings.

FIG. 1 depicts, according to an embodiment of the present invention, thenucleotide sequence of a 2.686 kb fragment (SEQ ID NO:1) of thejasmonate-induced protein gene (JAS) which lies immediately upstream ofthe putative translational start site. The putative TATA box, CAT boxsequence and first ATG are bolded.

FIG. 2 illustrates, according to an embodiment of the present invention,the JAS promoter/GUS expression vector (pJAS::GUSpUC19) suitable forexpression in sugarcane, maize and sorghum.

FIG. 3 illustrates, according to an embodiment of the present invention,the JAS promoter/GUS expression vector (pJAS::GUSpCambia) suitable forexpression in rice.

FIG. 4 is a DNA gel blot analysis of restriction digests of 36stem-regulated cDNA clones probed separately with cDNA from fivesugarcane tissues, stem (top, middle and bottom), leaf and root.

FIG. 5 is an image of a sugarcane cDNA microarray after hybridization.

FIG. 6 is a scatter plot representation of RNA expression levels of 251sugarcane cDNA clones as detected by the microarray analysis.

FIG. 7 graphically depicts the fold change in the RNA levels of threestem-regulated cDNAs, JAS, JAS and SSIP in stem vs leaf and stem vsroot, as evaluated by real-time quantitative RT-PCR.

FIG. 8 is an RNA gel blot analysis of the RNA levels of threestem-regulated cDNAs, JAS, JAS and SSIP in sugarcane leaf, stem androot.

FIG. 9 shows a DNA gel blot of several JAS positive genomic clones.

FIG. 10 shows the genomic walking protocol used to isolate the JASpromoter.

FIG. 11A shows a gel of the primary PCR products of a JAS clone. FIG.11B shows a gel of PCR products probed with a JAS oligo specific to the5′UTR of the JAS gene.

FIG. 12A shows sugarcane stems in which a JAS promoter has been used todrive expression of a GUS reporter protein. FIG. 12B is a close upshowing GUS expression in stem nodal areas and vascular tissue.

FIG. 13 shows a rice stem in which a JAS promoter has been used to driveexpression of a GUS reporter protein.

DETAILED DESCRIPTION

The present invention includes isolated nucleic acids having promoterscapable of directing specific expression in stem tissue or operable inresponse to stimulation by defense-inducing agents. In accordance withthe present invention, a subject promoter, when operably linked toeither the coding sequence of a gene or a sequence complementary to anative plant gene, directs expression of the coding sequence orcomplementary sequence in stem tissue or in response to adefense-inducing agent.

In one embodiment of the present invention, there is provided anisolated nucleic acid corresponding to a promoter isolated from asugarcane stem-regulated, defense-inducible gene, designatedjasmonate-induced protein (JAS) promoter, having the sequence ofnucleotides −2686 to −1 as depicted in FIG. 1 (SEQ ID NO:1).

The promoters of the present invention are useful in the construction ofexpression cassettes which include, in the 5′ to 3′ direction, apromoter which directs stem-regulated or defense-inducible expressionsuch as the JAS promoter, a heterologous gene or a coding sequence, orsequence complementary to a native plant gene under control of thepromoter, and a 3′ termination sequence. Such an expression cassette maybe incorporated into a variety of autonomously replicating vectors inorder to construct an expression vector.

In one embodiment of the present invention, there is provided a promoterfrom a sugarcane JAS gene. An isolated nucleic acid for a promoter froman JAS gene can be provided as follows. JAS recombinant genomic clonesare first isolated by screening a sugarcane genomic bacterial artificialchromosome (BAC) library with a cDNA (or a portion thereof) representingJAS mRNA. In order to obtain a cDNA representing JAS mRNA, a sugarcanestem-regulated cDNA library may be constructed and screened bydifferential hybridization with stem, leaf and root cDNA probes toidentify stem-regulated cDNAs including the JAS cDNA.

Methods considered useful in obtaining genomic recombinant DNA sequencescorresponding to the JAS gene by screening a genomic library areprovided in Sambrook et al. (1989), Molecular Cloning: A LaboratoryManual, Cold Spring Harbor, N.Y., for example, or any of the laboratorymanuals on recombinant DNA technology that are widely available.

To determine nucleotide sequences, a multitude of techniques areavailable and known to the ordinarily skilled artisan. For example,restriction fragments containing a corresponding JAS gene may besubcloned into the polylinker site of a sequencing vector such aspBluescript (Stratagene). These pBluescript subclones may then besequenced by the double-stranded dideoxy method (Chen et al. (1985) DNA,4; 165).

In a specific embodiment of the present invention, the JAS promoterincludes nucleotides −2686 to −1 of FIG. 1 (SEQ ID NO:1).

Modifications to the JAS promoter as set forth in SEQ ID NO:1, whichmaintain the characteristic property of directing stem-regulated ordefense-inducible expression, are within the scope of the presentinvention. Such modifications include insertions, deletions andsubstitutions of one or more nucleotides.

The subject JAS promoter may be derived from restriction endonuclease orexonuclease digestion of isolated JAS genomic clones. Thus, for example,the known nucleotide or amino acid sequence of the coding region of agene of the jasmonate-induced protein gene family is aligned to thenucleic acid or deduced amino acid sequence of an isolatedstem-regulated genomic clone and the 5′ flanking sequence (i.e.,sequence upstream from the translational start codon of the codingregion) of the isolated JAS genomic clone is located.

The JAS promoter as set forth in SEQ ID NO:1 (nucleotides −2686 to −1 ofFIG. 1) may be generated from genomic clones having either or bothexcess 5′ flanking sequence or coding sequence by exonucleaseIII-mediated deletion. This is accomplished by digesting appropriatelyprepared DNA with exonuclease III (exoIII) and removing aliquots atincreasing intervals of time during the digestion. The resultingsuccessively smaller fragments of DNA may be sequenced to determine theexact endpoint of the deletions. There are several commerciallyavailable systems which use exonuclease III (exoIII) to create such adeletion series, e.g. Promega Biotech, “Erase-A-Base®” system.Alternatively, PCR primers may be defined to allow direct amplificationof the subject JAS promoter.

Using the same methodologies, the ordinarily skilled artisan maygenerate one or more deletion fragments of the JAS promoter as set forthin SEQ ID NO:1. Any and all deletion fragments which include acontiguous portion of the nucleotide sequences set forth in SEQ ID NO:1and which retain the capacity to direct stem-regulated ordefense-inducible expression are contemplated by the present invention.

In addition to the sugarcane JAS promoter having the nucleotide sequenceset forth as −2686 to −1 in FIG. 1 (SEQ ID NO:1), the present inventionis directed to other promoter sequences which correspond to the samegene, i.e., a homolog, in other plant species. As defined herein, suchrelated sequences which direct stem-regulated or defense-inducibleexpression, may be described in terms of their percent homology oridentity on a nucleotide level to the nucleotide sequence (−2686 to −1)as set forth in FIG. 1 (SEQ ID NO:1). Alternatively, such relatedsequences from other plant species may be defined in terms of theirability to hybridize to the JAS promoter of SEQ ID NO: 1 under stringenthybridization conditions.

The present invention therefore contemplates nucleic acid sequenceshybridizing with the JAS nucleic acid sequence as set forth in FIG. 1(SEQ ID NO:1) and which differ in one or more positions in comparisonwith SEQ ID NO:1 so long as such hybridizing sequence corresponds to apromoter which directs stem-regulated or defense-inducible expression.

By “hybridizing” it is meant that such nucleic acid molecules hybridizeunder conventional hybridization conditions, preferably under stringentconditions such as described by, e.g., Sambrook (Molecular Cloning; ALaboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y. (1989)). An example of one such stringenthybridization condition is hybridization in 4×SSC at 65° C., followed bya washing in 0.1×SSC at 65° C. for one hour. Alternatively, an exemplarystringent hybridization condition is in 50% formamide, 4×SSC at 42° C.

Promoter sequences of the present invention may also be described interms of percent homology or identity on a nucleotide level to thenucleotide sequence −2686 to −1 depicted in FIG. 1 (SEQ ID NO:1). Thereare a number of computer programs that compare and align nucleic acidsequences which one skilled in the art may use for purposes ofdetermining sequence identity (sequence similarity).

Thus, an isolated nucleic acid is provided having a promoter whichdirects stem-regulated or defense-inducible expression has a sequenceidentity (sequence similarity) of about 60% to about 65% when comparedto the nucleotide sequence of the JAS promoter as set forth in SEQ IDNO:1. In a specific embodiment, an isolated nucleic acid including apromoter which directs stem-regulated expression has a sequence identity(sequence similarity) of about 65% to about 75% when compared to thesequence of the JAS promoter as set forth in SEQ ID NO:1.

In another specific embodiment, an isolated nucleic acid including apromoter which directs stem-regulated expression has a sequence identity(sequence similarity) of about 75% to about 85% when compared to thesequence of the JAS promoter as set forth in SEQ ID NO:1.

In another specific embodiment, an isolated nucleic acid including apromoter which directs stem-regulated expression has a sequence identity(sequence similarity) of about 85% to about 90% when compared to thesequence of the JAS promoter as set forth in SEQ ID NO:1.

In another specific embodiment, an isolated nucleic acid including apromoter which directs stem-regulated expression has a sequence identity(sequence similarity) of about 90% or greater when compared to thesequence of the JAS promoter as set forth in SEQ ID NO:1.

Sequences similar to a subject promoter may be identified by databasesearches using the promoter or elements thereof as the query sequenceusing the Gapped BLAST algorithm (Altschul et al., 1997 Nucl. Acids Res.25:3389–3402) with the BLOSUM62 Matrix, a gap cost of 11 and persistencecost of 1 per residue and an E value of 10. Two sequences may becompared with either ALIGN (Global alignment) or LALIGN (Local homologyalignment) in the FASTA suite of applications (Pearson and Lipman, 1988Proc. Nat. Acad. Sci. 85:2444–24448; Pearson, 1990 Methods in Enzymology183:63–98) with the BLOSUM50 matrix and gap penalties of −16, −4.

Nucleic acid molecules corresponding to promoters of the presentinvention may be obtained by using the subject 2,686 kb JAS promoter ofSEQ ID NO: 1 or a portion thereof (including fragments) or complementsthereof as a probe and hybridizing with a nucleic acid molecule(s) fromany higher plant. Nucleic acid molecules hybridizing to the 3.012 kb JASpromoter or a portion thereof can be isolated, e.g., from genomiclibraries by techniques well known in the art. Promoter fragmentshomologous to JAS may also be isolated by applying a nucleic acidamplification technique such as the polymerase chain reaction (PCR)using as primers oligonucleotides derived from sequence set forth in SEQID NO:1.

Confirmation of the stem-specificity or defense-inducibility of the JASpromoter (including modifications or deletion fragments thereof), andpromoters from homologous genes which direct stem-regulated ordefense-inducible expression, may be accomplished by construction oftranscriptional and/or translational fusions of specific sequences withthe coding sequences of a heterologous gene or coding sequence, transferof the expression cassette into an appropriate host, and detection ofthe expression of the heterologous gene or coding sequence. The assayused to detect expression depends upon the nature of the heterologousgene or coding sequence. For example, reporter genes, exemplified bychloramphenicol acetyl transferase and β-glucuronidase (GUS), arecommonly used to assess transcriptional and translational competence ofchimeric nucleic acids. Standard assays are available to sensitivelydetect the reporter enzyme in a transgenic organism.

The GUS gene is useful as a reporter of promoter activity in transgenicplants because of the high stability of the enzyme in plant cells, thelack of intrinsic GUS activity in higher plants and availability of aquantitative fluorimetric assay and a histochemical localizationtechnique. Jefferson et al. (1987) (EMBO J. 6: 3901–3907) haveestablished standard procedures for biochemical and histochemicaldetection of GUS activity in plant tissues. Biochemical assays areperformed by mixing plant tissue lysates with4-methylumbelliferyl-β-D-glucuronide, a fluorimetric substrate for GUS,incubating one hour at 37° C., and then measuring the fluorescence ofthe resulting 4-methyl-umbelliferone. Histochemical localization for GUSactivity is determined by incubating plant tissue samples in5-bromo-4-chloro-3-indolyl-glucuronide (X-Gluc) for about 18 hours at37° C. and observing the staining pattern of X-Gluc. The construction ofsuch expression cassettes allows definition of specific regulatorysequences and demonstrates that these sequences can direct expression ofheterologous genes, or coding sequences in a stem-regulated ordefense-inducible manner.

Another aspect of the invention is directed to expression cassettes andexpression vectors including a promoter which directs stem-regulated ordefense-inducible expression such as an JAS promoter or portion thereof,operably linked to the coding sequence of a heterologous gene such thatthe regulatory element is capable of controlling expression of theproduct encoded by the heterologous gene. The heterologous gene can beany gene other than JAS. If necessary, additional regulatory elementsfrom genes other than JAS or parts of such elements sufficient to causeexpression resulting in production of an effective amount of thepolypeptide encoded by the heterologous gene may be included in theexpression cassettes.

As used herein, the term “cassette” refers to a nucleotide sequencecapable of expressing a particular coding sequence if said codingsequence is inserted so as to be operably linked to one or moreregulatory regions present in the nucleotide sequence. Thus, forexample, the expression cassette may include a heterologous codingsequence which is desired to be expressed in a plant seed. Theexpression cassettes and expression vectors of the present invention aretherefore useful for directing stem-regulated or defense-inducibleexpression of any number of heterologous genes.

Accordingly, the present invention provides expression cassettesincluding sequences of a promoter which directs stem-regulated ordefense-inducible expression including the JAS promoter which areoperably linked to a heterologous gene such as a sugar transport gene orsugar accumulation gene. Examples of sugar transport genes useful forpracticing the present invention include sucrose transporters and othermonosaccharide and disaccharide transporters. Such sugar transportergenes have been isolated and characterized from sugarcane and otherplant species. Their nucleotide coding sequences as well as methods ofisolating such coding sequences are disclosed in the publishedliterature and are widely available to those of skill in the art.

Additionally, the present invention includes expression cassettes whichexpress products having an insecticidal or antimicrobial activity.Introduction into a plant cell of a expression cassette including aninsecticidal or antimicrobial gene operably linked to the subjectpromoter sequences such as the JAS promoter allows for protection in thestem. Examples of antimicrobial genes useful for practicing the presentinvention include chitinase and β-1,3-glucanase genes (Jach et al. 1995Plant Journal 8:97–109). Expression cassettes which include the subjectpromoter such as the JAS promoter operably linked to a bioinsecticidalpeptide or a defence elicitor peptide that upregulate the endogenouspathway are particularly contemplated to amplify natural defenseresponses.

The expression cassettes of the present invention may be constructed byligating a subject promoter such as the JAS promoter or part thereof tothe coding sequence of a heterologous gene. The juxtaposition of thesesequences may be accomplished in a variety of ways. In one embodiment,the sequence order is in a 5′ to 3′ direction and includes an JASpromoter and coding sequence of a heterologous gene. In another thesequence order in a 5′ to 3′ direction is an JAS promoter, a codingsequence and a termination sequence which includes a polyadenylationsite.

Standard techniques for construction of such expression cassettes arewell known to those of ordinary skill in the art and can be found inreferences such as Sambrook et al.(1989). A variety of strategies areavailable for ligating fragments of DNA, the choice of which depends onthe nature of the termini of the DNA fragments.

The restriction or deletion fragments that contain a subject promoterTATA box are ligated in a forward orientation to a promoterlessheterologous gene or coding sequence such as the coding sequence of GUS.The skilled artisan will recognize that promoters of the presentinvention and parts thereof, may be provided by other means, for examplechemical or enzymatic synthesis.

The 3′ end of a heterologous coding sequence is optionally ligated to atermination sequence including a polyadenylation site, exemplified by,but not limited to, the nopaline synthase polyadenylation site, or theoctopine T-DNA gene 7 polyadenylation site. Alternatively, thepolyadenylation site may be provided by the heterologous gene or codingsequence.

The present invention also provides methods of increasing expressionlevels of heterologous genes or coding sequences in plant stem tissues.In accordance with such methods, the expression cassettes and expressionvectors of the present invention may be introduced into a plant in orderto effect expression of a heterologous gene or coding sequence. Forexample, a method of producing a plant with increased levels of aproduct of a sucrose accumulating gene or a defense gene is provided bytransforming a plant cell with an expression vector including an JASpromoter or portion thereof, operably linked to a sucrose accumulatinggene or a defense gene and regenerating a plant with increased levels ofthe product of the sucrose accumulating gene or defense gene. In aspecific embodiment of the present invention, a transgenic sugarcaneline may be produced in which the sugar metabolism is altered toincrease its stem dry weight. This is of particular importance becausecommercial sugarcane varieties accumulate up to 50–60% of dry weight oftheir stem tissues as sucrose. In a further specific embodiment of thepresent invention, a transgenic sugarcane line may be produced withenhanced bioinsecticidal activity for protection against stem boringinsects, which are the most destructive pests. Expression of thebioinsecticidal protein may be induced in this case by the applicationof a defense inducing agent such jasmonic acid.

Another aspect of the present invention provides methods of reducinglevels of a product of a gene which is native to a plant which includestransforming a plant cell with an expression vector having an JASpromoter or part thereof, operably linked to a nucleic acid sequencewhich is complementary to the native plant gene. In this manner, levelsof endogenous product of the native plant gene are reduced through amechanism known as antisense regulation. Thus, for example, levels of aproduct of a sucrose accumulating gene or defense gene are reduced bytransforming a plant with an expression vector including an JAS promoteror part thereof, operably linked to a nucleic acid sequence which iscomplementary to a nucleic acid sequence coding for a native sucroseaccumulating gene or a defense gene.

The present invention also provides a method of silencing a gene nativeto a plant which includes transforming a plant cell with an RNAinterference (RNAi) expression vector including an JAS promoter or partthereof, operably linked to a nucleic acid sequence which is an invertedrepeat of the native plant gene. In this manner, levels of endogenousproduct of the native plant gene are reduced through a mechanism knownas RNA interference. Thus, for example, levels of a product of a sucrosesynthesis gene may be reduced by transforming a plant with an expressionvector including a subject stem-regulated promoter or part thereof,operably linked to a nucleic acid sequence which is an inverted repeatof the nucleic acid sequence coding for a native sucrose accumulatinggene or a defense gene.

The present invention further provides a method of cosuppressing a genewhich is native to a plant which includes transforming a plant cell withan expression vector having an JAS promoter or part thereof, operablylinked to a nucleic acid sequence coding for the native plant gene. Inthis manner, levels of endogenous product of the native plant gene arereduced through the mechanism known as cosuppression. Thus, for example,levels of a product of a sucrose synthesis gene or defense gene may beby transforming a plant with an expression vector including a subjectpromoter or part thereof, operably linked to a nucleic acid sequencecoding for a sucrose accumulating gene or defense gene native to theplant. Although the exact mechanism of cosuppression is not completelyunderstood, one skilled in the art is familiar with published worksreporting the experimental conditions and results associated withcosuppression (Napoli et al. (1990) The Plant Cell, 2; 270–289; Van derKrol (1990) Plant Mol. Biol, 14; 457–466.)

To provide regulated expression of the heterologous or native genes,plants may be transformed with the expression cassette constructions ofthe invention. Methods of nucleic acid transfer are well known in theart. The expression cassettes may be introduced into plants by leaf disktransformation-regeneration procedure as described by Horsch et al.(1985) Science, 227; 1229–1231. Other methods of transformation such asprotoplast culture (Horsch et al. (1984) Science, 223; 496; DeBlock etal. (1984) EMBO J., 2; 2143; Barton et al. (1983) Cell, 32; 1033) mayalso be used and are within the scope of this invention. In a specificembodiment, plants including rice and other monocots and dicots aretransformed with Agrobacterium-derived vectors such as the pCambiavectors described in Maliga et al. (1994) (Plant Mol. Biol., 35;989–994) (FIG. 3). In another specific embodiment, embryonic calli andplant organs are transformed by gene gun particle bombardment with theexpression vector shown in FIG. 2.

In another specific embodiment, stem tissues and embryonic calli areparticle bombarded using the gene gun/biolistic approach as described in(Klein et al. (1987) Nature, 327; 70). Other well-known methods areavailable to insert the expression cassettes of the present inventioninto plant cells. Such alternative methods include electroporation,chemically-induced DNA uptake, and use of viruses or pollen as vectors.

When necessary for the transformation method, the expression cassettesof the present invention may be inserted into a plant transformationvector, e.g. the binary vector described by Maliga et al. (1994) PlantMol. Biol., 25; 989–994. Plant transformation vectors may be derived bymodifying the natural gene transfer system of Agrobacterium tumefaciens.The natural system comprises large Ti (tumor-inducing)-plasmidscontaining a large segment, known as T-DNA, which is transferred totransformed plants. Another segment of the Ti plasmid, the vir region,is responsible for T-DNA transfer. The T-DNA region is bordered byterminal repeats. In the modified binary vectors, the tumor inducinggenes have been deleted and the functions of the vir region are utilizedto transfer foreign DNA bordered by the T-DNA border sequences. TheT-region also contains a selectable marker for antibiotic resistance,and a multiple cloning site for inserting sequences for transfer. Suchengineered strains are known as “disarmed” A. tumefaciens strains, andallow the efficient transfer of sequences bordered by the T-region intothe nuclear genome of plants.

Embryonic calli and and other susceptible tissues are inoculated withthe “disarmed” foreign DNA-containing A. tumefaciens, cultured for anumber of days, and then transferred to antibiotic-containing medium.Transformed shoots are then selected after rooting in medium containingthe appropriate antibiotic, and transferred to soil. Transgenic plantsare pollinated and seeds from these plants are collected and grown onantibiotic medium.

Expression of a heterologous or reporter gene in tissues, developingseeds, young seedlings and mature plants may be monitored byimmunological, histochemical, mRNA expression or activity assays. Asdiscussed herein, the choice of an assay for expression of theexpression cassette depends upon the nature of the heterologous codingsequence. For example, RNA gel blot analysis may be used to assesstranscription if appropriate nucleotide probes are available. Ifantibodies to the polypeptide encoded by the heterologous gene areavailable, Western analysis and immunohistochemical localization may beused to assess the production and localization of the polypeptide.Depending upon the heterologous gene, appropriate biochemical assays maybe used.

Another aspect of the present invention provides transgenic plants orprogeny of these plants containing the expression cassettes of theinvention. Both monocot and dicot plants are contemplated. Plant cellsmay be transformed with the expression cassettes by any of the planttransformation methods described above. The transformed plant cell,usually in the form of a callus culture, leaf disk, explant or wholeplant (via Agrobacterium-mediated transformation or gene gun particlebombardment) may be regenerated into a complete transgenic plant bymethods well-known to one of ordinary skill in the art (e.g., Horsh etal., 1985). In a specific embodiment, the transgenic plant is sugarcane,rice, maize, sorghum or other monocot plant. Because progeny oftransformed plants inherit the expression cassettes, seeds or cuttingsfrom the transformed plants may be used to maintain the transgenic line.

Finally, the invention includes methods of isolating a tissue-specificpromoter in polyploid monocots by first selecting potentialtissue-specific cDNA clones using differential hybridization analysisfollowed by microarray analysis if the cDNA clones using probes derivedfrom total RNA of various tissues of interest. Differentialhybridization use cDNA probes specific to tissue of interest. Thesetissues of interest may be selected to include not only a tissue inwhich expression is desired, but also other tissues in which expressionis not desired. This provides additional screening criteria.

This may be followed by further confirmation techniques such as realtime PCR and RNA gel blot analysis using total RNA of various tissues.Finally, because promoter/PCR walking is difficult in polyploid plants,the promoter may have to be isolated by screening a bacteriophagelibrary. If promoters thus located contain internal restriction sitesthat prevent removal from phage clones, genomic walking may be used toisolate the promoter.

EXAMPLES

The invention may be further understood through reference to thefollowing examples, which provide further description of specificembodiments of the invention. Variations of these examples will beapparent to one skilled in the art and are intended to be encompassed inthe present invention.

Example 1 Identification of Stem-Specific cDNAs

Stem-Specific cDNA Macroarray Analysis

To identify cDNAs that were expressed in sugarcane stems, a cDNA libraryrepresenting stem mRNA was constructed and screened by differentialhybridization.

In order to have a cDNA library that has a good representation of mRNAfrom all regions of the stem, mRNA was separately isolated from top, midand bottom portions of the stem of Saccharum spp. cultivar CP72-1210 anda pooled sample was used for preparation of the cDNA. Total RNAs andpoly(A)+ RNAs were prepared using the RNeasy kit and Oligotex kit fromQiagen, respectively. For the synthesis of cDNAs, the SMART PCR cDNAlibrary construction kit (BDBiosciences Clontech) was selected sincethis technology utilizes a unique SMART oligonucleotide (cap finder) inthe first strand cDNA synthesis followed by a long distance PCR (LD PCR)amplification to generate high yields of full-length ds cDNAs. A pooledsample consisting of 100 ng of each of stem top, mid and bottom poly(A)+RNAs was used for the preparation of first strand cDNAs which were thenselected to synthesize ds cDNAs by LD PCR. The sizes of ds cDNA rangedfrom 300 bp to 6 kb. To eliminate small and partial (less thanfull-length) cDNAs, the ds cDNA sample was subjected to gelfractionation and the agarose gel slice containing 500 bp to 3 kb wasexcised and purified. This selected cDNA fraction was cloned into pCR2.1vector (TA cloning kit, Invitrogen) using DH10B Escherichia colibacterial cells for transformation. The ampicillin-resistant and whitecolonies were picked and archived in 384-well microtiter plates. A totalof 13,824 cDNA clones (archived in thirty six 384-well microtiterplates) were obtained. About 100 clones were randomly picked, and DNApreparations were made and tested by restriction analysis to revealinsert sizes of 300 bp to 1500 bp.

Next the cDNA library was screened using differential hybridization toide□tify stem-specific cDNAs. Specifically, macroarrays consisting highdensity replica filters (three copies: A, B and C) of the library wereprepared using a 3×3 duplicate grid pattern using a Beckman Biomek 2000.Each copy of the library (9 filters) was first probed withradioactively-labeled (random decamer priming method; Decaprime II kit,Ambion) cDNA of top, mid and bottom stem tissues, respectively.Hybridization were carried out at 65.degree.C. and blots were washed upto the final stringency of 0.3.times.SSC/0.1.percen.SDS and exposed toX-ray films. After the autorads from these hybridizations were obtained,filters of copies A and B were stripped and hybridized with 32P-labeledcDNA probes of leaf and root tissues, respectively. Both strong and weakhybridization signals were recorded with an intention of identifyingcDNAs that are strongly expressed in one type of tissue but weak in allothers and vice versa. Using this strategy, information from allpossible combinations (stem top vs root, stem mid vs leaf, stem mid vsroot, stem bottom vs leaf, stem bottom vs root, stem top vs stem mid,stem top vs stem bottom, stem mid vs stem bottom and leaf vs root) wasgathered. Data obtained from these substractions revealed that a totalof 188 cDNAs were present specifically in stem and 41 cDNAs displayedconstitutive expression. To further corroborate the expression patternsof these cDNA clones, DNA gel blots were prepared using restrictiondigests of 36 selected clones and probed with radioactively-labeled cDNAfrom five tissues separately. Based on these analyses, 25 clonesdisplayed stem-specific expression and 11 cones showed constitutiveexpression (See FIG. 4).

Stem-Specific cDNA Microarray Analysis

The macroarray analysis of the potential stem-specific CAN clones wasfollowed by a focused microarray analyis. A DNA microarray including thepanel of the 188 stem-specific cDNA clones initially identified by themacroarray analysis along with 63 additional cDNA clones was constructedto provide a sensitive assessment of their differential RNA expressionprofiles. Amplified PCR products for the 251 cDNAs were printed induplicate on glass microscope slides using a gridding robot fromGenemachines. The resulting microarray was probed with fluorescentlylabeled cDNAs synthesized from 1.mu.g of total amplified RNA (MessageAmpaRNA kit from Ambion for RNA amplification). One cDNA probe (e.g. stemRNA) is labeled with one dye (Cy5), and the second (e.g. leaf or rootRNA) with another dye (Cy3) using the 3DNA Array 350RP expression arraydetection kit of Genisphere and based on the two-color fluorescenthybridization microarray system developed by Brown and colleagues atStanford (Derisi et al. 1996 Science 278:680–686). The two probeslabeled with distinct dyes were hybridized simultaneously to a singleDNA array to allow for comparison of two different RNA populations orinternal standardization. Hybridization was detected and quantified witha confocal laser scanner (Affynetrix) that captures the image of theemission wavelength of each dye. A combined image of the microarrayhybridized with fluorescent probes representing stem RNA and leaf RNA isshown in FIG. 5. The resulting digital data was analyzed with theGenePix analysis software (Axon) and GeneSpring analysis software(Silicon Genetics). FIG. 6 shows a scatter plot representation of theRNA expression levels of the 251 cDNA clones of the microarray afterhybridization with probes from stem and leaf RNAs. Each ‘+’ symbolrepresents a cDNA. The normalized abundance of each cDNA derived fromstem RNA (Cy5) (y axis) was plotted vs that derived from leaf RNA (Cy3)(x axis). Data were normalized using the endogenous constitutive geneubiquitin. The central magenta line indicates equal hybridization ofboth Cy5 and Cy3 probes. The outer magenta line indicates expressionlevels of genes that are two-fold up or down.

Among the 251 cDNAs examined in the microarray analysis, 41 appeared tobe at least two-fold upregulated in the stem, whereas 14 wereconstitutively expressed.

These 41 cDNAs were sequenced using cycle sequencing with an ABI PRISMdye terminator cycle sequencing kit. Database searches for homologousnucleic acids were performed through NCBI using the BLAST algorithm.Several of the 41 stem-upregulated cDNA clones were found to belong tomultigene families. One cDNA that exhibited a 3.2-fold upregulationshared significant sequence similarity to a monocot gene involved indefense pathways o-methyl transferase (OMT). In maize OMT is a keyenzyme in the biosynthetic pathway of phenolic compounds. It is inducedby fungal pathogens and other stress conditions and functions in stresscompensation and lignification (See Held, B. M. et al., Plant Physiol.,102:1001–1008, 1993.)

The expression levels of other cDNAs were also found to be two-foldupregulated. These include the cDNAs for jasmonate-induced protein(JAS). (See Lee, J. Plants, 199:625–632, 1999.) Other upregulated cDNAwere found to correlate with additional genes, such as maize o-methyltransferase (OMT) gene and the salt stress-induced protein gene (SSIP).Upregulation findings are presented in Table 1.

TABLE 1 Cy5/Cy3 Ratios (Upregulation) of Stem- regulated Genes inMicroarray Analysis Stem-Upregulated Genes Normalized Cy5/Cy3 Ratios JASClone 6H10 9.1 JAS Clone 20G16 4.7 JAS Clone 5P22 4.6 JAS Clone 20F183.6 JAS Clone 17C11 3.2 JAS Clone 4O3 3.1 JAS Clone 24G4 3.1 JAS Clone10P7 3.0 JAS 3.2 SSIP 5.2 Protein translation factor 3.0 SUI1 homologReal-Time Quantitative RT-PCR Analysis of Gene Expression in Stem

The expression patterns of four selected cDNAs that were upregulated atleast two-fold in the microarray analysis were further assessed byreal-time quantitative PCR (RT-PCR). The four cDNAs include OMT, JASclone 20F18, JAS clone 6H10 and SSIP. The real-time RT-PCR approachallows the identification of false positives obtained through themicroarray analysis as well as the differentiation between members ofmultigene families.

Total RNA (2.mu.g) was extracted from sugarcane stem, leaf or root andthe corresponding cDNA templates were generated through RT-PCR using theTaqMan reverse transcription kit (Applied Biosystems). PCR was thenperformed using the cDNA templates and primers that are specific to thecDNAs of OMT, JAS, SSIP and ubiquitin (endogenous control). Primers weredesigned by the Primer Express software (Applied Biosystems) and labeledwith the fluorescent SYBR Green 1 dye that binds to double-stranded DNA(SYBR Green PCR master mix; Applied Biosystems). The increase influorescence was measured for each PCR cycle with the ABI Prism 7700Sequence Detection System (Applied Biosystems) since the incorporationof SYBR Green 1 dye into a real-time PCR reaction allows the detectionof any double-stranded DNA generated during PCR. Real-time RT-PCRquantitations (Ct values) for the four cDNAs as normalized to theendogenous constitutive control ubiquitin are shown in FIG. 7. Theseresults demonstrate that OMT as well as SSIP and the two JAS clones arestrongly expressed in stem as compared to leaf or root.

Quantitative RNA Gel Blot Analysis of Gene Expression in Stem

To further confirm actual upregulation of OMT in sugarcane stem, aquantitative RNA gel blot analysis was performed using total RNA(12.mu.g) from stem, leaf or root and radioactive probes specific toOMT, SSIP, JAS clone 20F18 and ubiquitin. Hybridization signals wererecorded with a Fujix BAS 2000 phosphoimager. The data were analyzedusing MacBas software. The hybridization signal for each sample wasquantitated and adjusted for loading by virtue of hybridization to aubiquitin cDNA probe. The quantitative accumulation of OMT, JAS and SSIPwas determined and compared to that of the constitutive ubiquitin gene(See FIG. 8). All 3 cDNAs were highly expressed only in stem.

JAS upregulation as confirmed by the three analytical methods describedabove is summarized in Table 2.

TABLE 2 Upregulation of JAS and SSIP in Stem Tissue Microarray foldRT-PCR fold RNA gel blot increase change fold change (stem v. Stem v.Stem v. Stem v. Stem v. Gene leaf) leaf root leaf root JAS 3.6 1.6 1.31.3 1.4 SSIP 5.2 1.0 1.0 4.5 5.7

Example 2 Effects of Defense-Inducing Agents on OMT Upregulation

The 251 cDNA clones used for microarray analysis in Example 1 werefurther used to assess the effects of defense-inducing agents on theexpression of the stem-specific genes.

Total RNA was extracted from wild type sugarcane plants (3 months-old)that were sprayed with the defense-inducing agents jasmonic acid (JA)(25.mu.M in 0.05.percent.Tween 20), methyl-jasmonate (MeJA) (200.mu.M in0.1.percent.ethanol and 0.05.percent.Tween 20) or salicylic acid (SA) (5mM in 0.05.percent.Tween 20) for two time periods, 24 and 48 hours. Themicroarray including 251 cDNAs was probed with fluorescently labeledcDNAs synthesized from 1.mu.g of total amplified RNA. One cDNA probe(e.g. RNA from plant treated with a defense-inducing agent) is labeledwith one dye (Cy5), and the second (e.g. RNA from untreated controlplant) with another dye (Cy3)

Microarray data was analyzed using GeneSpring analysis software. Datawere normalized using an endogenous constitutive gene. The normalizedratio of Cy5 v. Cy3 for JAS intensity after treatment with eachdefense-inducing agent is summarized in Table 3.

TABLE 3 Cy5/Cy3 Ratios (Upregulation) for JAS Following Treatment withDefense-Inducing Agents JA Treatment MeJA Treatment SA treatment Clone24 hours 48 hours 24 hours 48 hours 48 hours 13F16 0.51 2.21 0.25 0.34No data 6H10 0.42 2.19 0.15 0.19 19.22 17C11 0.35 2.12 0.17 0.17 18.834Q3 0.41 2.12 0.15 0.18 15.26 5P22 0.41 2.10 0.16 0.17 18.08 10P7 0.592.10 0.13 0.15 20.96 2I1 0.59 2.10 0.27 0.22 16.92 20F18 0.34 2.00 0.160.18 19.54 12F18 0.51 1.64 0.20 0.21 17.54 20G16 0.85 1.30 0.50 0.4913.81 24G4 0.41 1.14 0.37 0.41 7.73 25K14 1.73 1.65 3.07 1.02 0.70 18H161.90 1.40 3.00 0.79 0.70

These results generally show that the JAS gene is upregulated byapproximately 48 hours after treatment with a defense-inducing agent.

Example 3 Isolation and Cloning of Genomic Sequence Corresponding to JASGene and Promoter Release

Initially, an attempt was made to use PCR walking to clone the JASpromoter directly from genomic DNA. However, the complexity of thesugarcane genome and the presence of substantial amounts of repetitiveDNA in it rendered this approach unworkable.

A second attempt to locate the JAS promoter was made using a sugarcanebacteriophage lambda (BAC) genomic library. BAC library filters wereprobed using a full-length JAS cDNA probe. Three screening wereperformed to select genomic clones exhibiting strong hybridization toJAS cDNA. Phage DNA was isolated from 13 candidate JAS genomic clonesand digested with restriction endonucleases (EcORI and HindIII) prior toDNA gel blot analysis. DNA gel blot analysis (shown in FIG. 9) revealedcommon restriction fragments containing the JAS gene. This indicatedthat the 13 JAS clones were most likely representative of a single JASlocus.

However, the JAS promoter could not be directly released from any BACclone because of the presence of several restriction fragments withineach clone. Genomic walking was required to obtain the promoter. Using aClontech Genome Walker kit, the JAS gene was amplified from libraries.The general PCR-based approach is shown in FIG. 10. FIG. 11A shows a gelof the primary PCR products of the JAS BAC clone 159E16. PCR wasconducted using an adapter primer and a 3′ primer specific to the JAS 5′UTR. FIG. 11B shows a gel blot of PCR products probed using a JAS oligospecific to the 5′UTR of JAS.

The JAS promoter was thus identified and sequenced to reveal a 2.686 kbpromoter fragment (See FIG. 1 and SEQ ID NO:1.)

Example 4 Vector Construction and Expression in Planta

A sugarcane expression vector was produced by cloning either the JASpromoter into a pUC19 GUS reporter vector to produce the pJAS::GUSpUC19vector. (See FIG. 2.) Specifically, the 35S promoter normally found inpUC19 GUS was replaced with a JAS promoter at the corresponding XbaI andNcoI restriction sites to create a translational fusion with the GUSreporter DNA.

A rice expression vector was also produced by cloning the pJAS promoterinto the plant binary expression vector pCambia 1301 to createpJAS::GUSpCambia. (See FIG. 3.) The 35S promoter normally pCambia 1301was replaced at the corresponding XbaI and NcoI restriction sites tocreate a translational fusion with the GUS reporter DNA.

Stable expression of GUS under control of the JAS promoter was achievedin sugarcane. Furthermore, such expression was stem-specific.

Specifically, embryonic calli of sugarcane cultivar CP72-1210 weretransformed with pOMT::GUSpUC vectors using gene gun/biolistic-mediatedtransfer (Irvine and Mirkov, 1997 Sugar Journal 60:25–29; Inglebrecht etal, 1999 Plant Physiol. 119;1187–1197). Histochemical localization ofGUS expression was determined using 5-bromo-4-chloro-3-indoylglucuronide. (See FIG. 12A.) Intense GUS staining was observed in thestem nodal area and vascular bundles. (See FIG. 12B.)

Embryonic rice calli (Taipei 309) were transformed with pOMT:pUCpCambiausing Agrobacterium tumefaciens EHA 105 (Aldemita and Hodges 1996).Histochemical analysis of transformed clones showed intense GUS stainingin the stem vascular bundles. (See FIG. 13.)

Tissue analysis was used to confirm the presence of GUS in transformedplants. Expression as compared to that driven by maize ubiquitin 1 wasalso studied. The results of this analysis are presented in Table 4.

TABLE 4 Evaluation of JAS Promoter Activity in Rice and Comparison toUbiquitin 1 Promoter Tissue Line Stem Leaf Root Un- 0.19 ± 0.01  0.1 ±0.001 0.44 ± 0.11 trans- form- ed pJAS:: GUS #4 540.51 ± 119.08 248.34 ±16.39  253.67 ± 24.83  #6 10.28 ± 0.67  0.41 ± 0.02 2.31 ± 0.30 #8 21.29± 3.77  0.06 ± 0.03 12.5 ± 2.83 #23 551.13 ± 70.71    338 ± 21.36 320.01± 22.36  pUbi1:: GUS #5 32.50 ± 1.38  1.51 ± 0.21 7.22 ± 0.28 #12 181.12± 30.40  1043.88 ± 142.25  1641.76 ± 98.16  #13 1418.22 ± 408.17  337.25± 88.43  158.60 ± 10.43 Values expressed are pmole 4-methylumbelliferyl β-D-glucuronide/μg totalprotein/minute. All assays were performed in triplicate on 2 separatebatches of samples.

1. An isolated nucleic acid comprising a promoter having a sequence ofSEQ ID NO:1, wherein the promoter has stem-specific promoter activity.2. An isolated nucleic acid comprising a JAS promoter having a sequenceof SEQ. ID. NO. 1 and an exogenous nucleic acid, wherein the JASpromoter is operable to drive stem-specific expression or transcriptionof the exogenous nucleic acid.
 3. The nucleic acid of claim 2, whereinthe JAS promoter is further operable to drive upregulated stem-specificexpression or transcription in the presence of a defense-inducing agent.4. An expression vector comprising, in a 5′ to 3′ direction: a JASpromoter having a sequence of SEQ. ID. NO. 1; an exogenous nucleic acid;and a 3′ termination sequence, wherein the JAS promoter hasstem-specific promoter activity.
 5. The expression vector of claim 4,wherein the exogenous nucleic acid comprises a transgene.
 6. A bacterialcell comprising an expression vector having: a JAS promoter having asequence of SEQ. ID. NO. 1; an exogenous nucleic acid; and a 3′termination sequence, wherein the JAS promoter has stem-specificpromoter activity.
 7. An isolated nucleic acid comprising a fragment ofsequence of SEQ ID NO:1, wherein said fragment has stem-specificpromoter activity.
 8. An isolated nucleic acid comprising a fragment ofsequence of SEQ ID NO:1 and an exogenous nucleic acid, wherein saidfragment has stem-specific promoter activity and wherein said fragmentis operable to drive stem-specific expression or transcription of theexogenous nucleic acid.
 9. The nucleic acid of claim 8, wherein the JASpromoter is further operable to drive upregulated stem-specificexpression or transcription in the presence of a defense-inducing agent.10. An expression vector comprising, in a 5′ to 3′ direction: a JASpromoter having a fragment of sequence of SEQ ID NO:1 which is capableof stem-specific promoter activity; an exogenous nucleic acid; and a 3′termination sequence, wherein the JAS promoter has stem-specificpromoter activity.
 11. The expression vector of claim 10, wherein theexogenous nucleic acid comprises a transgene.
 12. The expression vectorof claim 10, wherein the expression vector is located in a bacterialcell.